Fluorescence methods are widespread in chemistry and biology. The methods give useful information on structure, distance, orientation, complexation, and location for biomolecules. Fluorescence based sensors provide sensitive means of determining the presence of compounds of interest in a sample. Various signal transduction methodologies can be use in measuring the response, where the sensor can produce a detectable change in fluorescence upon interacting with an analyte. Fluorescent sensors can provide desirable properties such as water solubility, low detection limits, and high selectivity for a desired analyte, where the analyte can be a small molecule, an enzyme, a catalytic metal, and the like.
It is clear that there is more than one type of interaction between light-absorbing molecules. One useful class of interaction is Forster energy transfer, also called fluorescence resonance energy transfer, or FRET. In this interaction, fluorescence excitation energy is transferred from a donor to an acceptor fluorophore. The extent of transfer depends on distance and on overlap in emission and absorption of donor and acceptor. FRET can occur over relatively long distances (tens of Angstroms). A second form of energy transfer is exciplex formation, which involves bonding between an excited-state fluorophore and a neighboring ground-state fluorophore, yielding a delocalized excited state. This results in a long red shift to fluorescence. Exciplexes can form only between molecules in direct contact or very nearly so. Exciplexes between two of the same molecules are known as excimers. Another class of interaction involving a fluorophore is quenching, in which a molecule causes the quantum yield of nearby fluorescent molecule to be lowered.
Chemists have in the past 10-20 years developed molecular sensors for specific chemical species. Fluorescent sensors have been developed that function in identifying and quantitating individual metal cations (such as Cd2+ or Hg2+)2,3 at low micromolar, and sometimes nanomolar, concentrations. Fewer examples exist for fluorescence sensing of toxic anions, although there are a few recent reports of sensors for cyanide and peroxynitrite.4,5 Chemists and engineers have also worked recently on “artificial nose”/“artificial tongue” designs, where organic vapors are adsorbed by polymers or groups of compounds, and result in fluorescence quenching, or in color changes in nonfluorescent materials. Such approaches have worked for specific species, such as polynitrotoluenes from explosives, and have been used for distinguishing organics in soft drinks. For example, sensors find use in remediation of industrial work and storage sites. At such sites frequent monitoring is required in addressing spills, leakage and contamination, and restoring the sites to a clean condition, to minimize environmental and human exposure.
Despite this previous work, there is no general fluorescent sensor design that can sense a wide variety of species such as cations, anions, vapors, gases, and neutral organics. Moreover, making sensor molecules is usually a laborious process involving multiple steps of organic synthesis by hand. In addition, the use of different traditional sensors would require different excitation wavelengths and filters. The present invention addresses all of these needs, by providing oligomeric sensor molecules that are easily constructed on an automated synthesizer. The large diversity of these molecules allows for specific sequences that yield a signal for a wide variety of molecular species; moreover, many of the molecules can be excited by one wavelength of light.
The present invention provides nucleoside analogs and sensors incorporating the subject nucleoside analogs. Combinatorial sequences of fluorophores built on a nucleic acid backbone may be generated and screened to identify sensors of molecular species with suitable fluorescent properties. The present invention provides sensors for simultaneous multiplexed detection of several molecular species.
Related Publications
Kool, U.S. Pat. Nos. 6,670,193 and 6,479,650, disclose fluorescent nucleoside analogs and combinatorial fluorophore arrays comprising same.
Kool et al., J. Am. Chem. Soc. 2006, 128, 6164-6171 disclose the sensing of metal ions with DNA building blocks such as fluorescent pyridobenzimidazole nucleosides.
Ellington, US Application 20050106594, disclose methods of selecting aptamer beacons in vitro using single-stranded nucleic acid species comprising a fluorphore and a random region of N nucleotides.
White et al., 2008 “Solid-State, Dye-Labeled DNA Detects Volatile Compounds in the Vapor Phase,” PLoS Biol 6(1): e9. doi:10.1371/journal.pbio.0060009 disclose the detection of odors by changes in fluorescence of dye-labeled, solid-state DNA dried onto a surface. Suslick et al., Anal Chem. 2006, 78(11):3591-600 disclose a colorimetric sensor array of chemoresponsive dyes for the detection and identification of volatile organic compounds (VOCs). Freud and Lewis, PNAS 92, pp. 2652-2656, 1995 disclose a chemically diverse conducting polymer-based ‘electronic nose’ sensitive to the identity and concentration of various vapors in air.